We are working to understand the molecular basis of our finding that propagation of different yeast prions is affected in dramatically different ways by the 98% identical and functionally interchangeable Hsp70 isoforms Ssa1 and Ssa2. We are also assessing the extent that stress-inducible Hsp70 counterparts Ssa3 and Ssa4, which are 88% identical to each other and 80% identical to Ssa1/2, influence propagation of different yeast prions. We earlier constructed a yeast system to evaluate function of any Hsp70 isoform in cell growth and prion propagation. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. Using it we showed that naturally occurring Hsp70 isoforms influence propagation of different yeast prions in different ways. We find PSI prion propagation was normal in cells expressing Ssa1 as the only cytosolic SSA Hsp70 but was antagonized in cells expressing only Ssa2 or Ssa4. PSI propagation was actually more robust in cells expressing only Ssa3. In comparing effects of these chaperones on URE3 prion propagation we saw opposite effects. URE3 propagated better in cells expressing only Ssa2 or Ssa4 and was weaker in cells expressing Ssa1 or Ssa3. We further showed that cells lacking both Ssa1 and Ssa2 were immune to infection by URE3 but not PSI. As for all infectious elements, critical processes for prion propagation are growth and replication. We are investigating our hypothesis that the different prions grow and replicate at different rates and that some Hsp70 isoforms affect growth more than replication, and others affect replication more than growth.[unreadable] Our genetic studies of prion-impairing Hsp70 mutants point to a common alteration in the Hsp70 reaction cycle (disproportionate time in ADP-bound state) as the basis for the anti-prion effects. We have undertaken biochemical measurements of specific enzymatic activities such as ATP hydrolysis, ADP exchange, substrate binding and protein chaperone function of purified wild type and mutant Ssa proteins. Our data confirm the conclusion from our genetic studies and show that regulation of Hsp70 activity, rather than direct substrate interactions, has the most influence on prions. This biochemical work further showed that this regulation can be disrupted in many different ways to inhibit prions.